The metabolic disposition of (14C) warfarin was studied in germ-free and acclimatized germ-free rats. During the 48 hr after the intraperitoneal administration, the germ-free rats excreted approximately a two-fold lower amount of the administered dose in the urine than the acclimatized rats. In contrast, the germ-free rats excreted approximately a two-fold larger amount of the administered dose in the feces than the acclimatized rats. These results indicate that the gut flora are involved in the metabolism and disposition of warfarin. They appear to affect the enterohepatic recycling of warfarin and its metabolites by hydrolyzing glucuronide and possibly sulfate conjugates.